Brain Research 940 (2002) 49–54 www.elsevier.com / locate / bres
Research report
Alzheimer’s disease amyloid beta and prion protein amyloidogenic peptides promote macrophage survival, DNA synthesis and enhanced proliferative response to CSF-1 (M-CSF) John A. Hamilton a , *, Genevieve Whitty a , Anthony R. White b,c , Michael F. Jobling b,c , Andrew Thompson d , Colin J. Barrow d , Roberto Cappai b,c , Konrad Beyreuther e , Colin L. Masters b,c a
Arthritis and Inflammation Research Centre, The University of Melbourne, Department of Medicine, The Royal Melbourne Hospital, Clinical Sciences Building, Royal Parade, Parkville, Victoria 3050, Australia b Department of Pathology, The University of Melbourne, Melbourne, Victoria 3010, Australia c The Mental Health Research Institute, Parkville, Victoria 3052, Australia d School of Chemistry, The University of Melbourne, Melbourne, Victoria 3010, Australia e Centre for Molecular Biology, The University of Heidelberg, IM Neuenheimer Feld 282, 69120 Heidelberg, Germany Accepted 14 February 2002
Abstract Microglial cells, macrophage-lineage cells in the brain, are increased in amyloid-containing plaques in Alzheimer’s disease (AD) and in the lesions of prion diseases. Recent studies suggest that microglia have a central role in turnover of amyloid in these diseases. We report here that synthetic amyloid beta (Ab) 1-42 and prion protein (PrP) 106-126 peptides promote macrophage survival; they also induce macrophage DNA synthesis, particularly in the presence of sub-optimal concentrations of the growth factor, macrophage-colony stimulating factor (M-CSF or CSF-1). These responses are proposed to provide a means to increase brain microglia / macrophage numbers thereby enhancing subsequent inflammatory / immune responses. These fibrillogenic peptides join the list of aggregates having these effects on macrophages, indicating the generality of this type of response. 2002 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Glia and other non-neuronal cells Keywords: Ab peptide; Prion protein; Macrophage; M-CSF (CSF-1); Survival; DNA synthesis
1. Introduction Alzheimer’s disease (AD) is a progressive, neurodegenerative disease characterized by the presence of numerous amyloid plaques and neurofibrillary tangles. The plaques and cerebral neuropil feature reactive microglia; the major component of plaques is the Ab peptide, a fragment of a larger, membrane spanning glycoprotein, the amyloid precursor protein. The predominant forms of Ab in AD lesions are fragments at residues 42 or 40. There is
*Corresponding author. Tel.: 161-3-8344-5480; fax: 161-3-93471863. E-mail address:
[email protected] (J.A. Hamilton).
substantial evidence that progressive cerebral accumulation of Ab is an early and perhaps necessary feature of the sporadic forms of AD. One pathway of Ab-induced neuron damage may involve inflammatory cells such as macrophages and reactive microglia. The latter cells are associated with most types of amyloid deposition and are increased in regions around compact amyloid deposits where they surround and infiltrate into the Ab plaques [36]. The functions of this macrophage-lineage cell in AD are not known, although they have been considered as plaque-attacking scavenger cells, as sources of cytokines and other inflammatory mediators, and even as producers of Ab [12]. Conversely, these cells may mediate the degradation and clearance of Ab from the extracellular space. This pathway may underlie the beneficial effects of
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immunization in experimental models of Ab cerebral deposition [31]. Associated with the transmissable spongiform encephalopathies (prion diseases) is the conversion of a normal cellular glycoprotein, the prion protein (PrP c), to an abnormal isoform, the scrapie isoform (PrP Sc), which shows increased protease resistance and insolubility and which accumulates in affected individuals, often in the form of extracellular amyloid deposits [29]. In addition to these properties shared with Ab, PrP Sc likewise has a high tendency to aggregate into fibrils [29]. PrP 106-126, a synthetic peptide corresponding to amino acid residues 106–126 of human PrP, is used as a model of prion protein neurotoxicity, forms amyloid-like fibrils in vitro, and can cause microglial cell activation and proliferation [5]. There are mouse data indicating that adult brain macrophages and a subpopulation of microglial cells are of bone marrow origin [8]. Macrophage-colony stimulating factor (M-CSF or CSF-1) is an important regulator of macrophage lineage development and function throughout the body [4]. There are in vivo and in vitro data showing that CSF-1 is important for normal microglial development as well as having a role in the cascade of events which surround the neurodegeneration in AD [1,9,11,24,39]. Macrophages are widely used as a model to study microglial activation by the amyloidogenic fragments of APP and PrP (see for example Ref. [6]). Involvement of scavenger receptors has been suggested for Ab microaggregate uptake [26]. The engorgement of microglia with undigested Ab is strikingly similar to the conversion of macrophages into foam cells when excessive amounts of cholesterol are taken up and stored as cholesterol ester droplets [25]. We have recently found that poorly degraded particulates, such as oxidized LDL, acetylated LDL, certain adjuvants (for example, alum) and arthritogenic crystals, promoted macrophage survival and DNA synthesis, and also allowed the loaded cells to proliferate strongly to low doses of CSF-1 [13–16]. Given the increasingly important role of microglia / macrophages in amyloid turnover and clearance in neurodegenerative diseases, we sought to determine whether Ab and PrP would behave similarly to these particulates.
2. Material and methods
2.1. Cells Bone marrow cells were obtained from male or female CBA mice as described before [13,33,34]. Bone marrowderived macrophages (BMM) were generated as adherent cells from progenitors in bone marrow and grown to confluence in 24-well plates (Nunc) in 20% L-cell conditioned medium (a crude source of CSF-1) [13,34]. The
BMM are a pure macrophage population with $95% capable of proliferation in response to CSF-1 [33,34]. Cells were prepared for experiments by washing twice with PBS and the experiment was either commenced immediately or the cells incubated 24 h in growth medium without L-cell conditioned medium (CSF-1-deprived) to render them quiescent before commencement [13,33,34].
2.2. Cell number and DNA synthesis For quantification of the number of BMM, viable cells were counted in a hemocytometer (trypan blue exclusion) [13–16]. DNA synthesis was measured as the incorporation of [methyl- 3 H]thymidine (TdR) (2 mCi / ml) [13– 16,34].
2.3. Reagents Ab1-42 was purchased from the Keck Laboratory, Yale University or Biopeptide Co., San Diego, CA; Ab42-1 was from Biopeptide Co. Other peptides were prepared as follows: Ab1-40 [17], PrP106-126 and scrambled PrP106126 [20] and PrP178-193 [32]. HPLC purified peptides were dissolved in sterile, pyrogen-free, distilled H 2 O at a concentration of 1 mg / ml and diluted to 500 mg / ml in RPMI medium prior to use. All of the peptides used in this study had been stored $2 weeks at 4 8C prior to use. The following reagents were obtained from commercial sources: [ 3 H]TdR and FCS [13–16,34]. Recombinant human CSF-1 (M-CSF) was a gift from Chiron, Emeryville, CA, USA. All practical precautions for minimizing endotoxin contamination were taken [13–16,34].
3. Results
3.1. Effect of Ab and PrP peptides on macrophage survival BMM are all phagocytic, macrophage lineage cells since they all depend on CSF-1 for their survival and proliferation and are widely used to study CSF-1 signaling [19,22,33–35]; when deprived of CSF-1 they all gradually detach from the tissue culture surface and die [19,33]. As for other hemopoietic cells, this death is by apoptosis (see, for example, Ref. [38]). When Ab1-42 or PrP106-126 was added to BMM in the absence of CSF-1 the cells remained spread on the tissue culture surface and survived better than untreated cells. In Fig. 1, for the experiment whose data are shown, it can be seen that 10 5 plated BMM are reduced to 3.5310 4 cells over a 3-day period following CSF-1 removal; Ab 1-42 (11 mM) and PrP106-126 (26 mM) reduced this loss to 7.5310 4 and 6.1310 4 cells, respectively. This effect was dose-dependent, occurring approximately between 12.5 and 100 mg / ml (3 and 22
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Table 1 Effect of Ab and PrP on BMM DNA synthesis Addition
[ 3 H]TdR incorporation (cpm310 23 )
– Ab 1-42 PrP178-193 CSF-1
12.660.5 42.660.9 55.460.8 198619
BMM, from which CSF-1 had just been removed, were cultured for 3 days in RPMI / 10% FBS in the absence or presence of 50 mg / ml Ab1-42 or PrP178-193; CSF-1 (5000 U / ml) was included as a separate positive control group. Cultures were pulsed with [ 3 H]TdR at 24 h for a further 48 h before harvest. Data are mean values from triplicate cultures from a representative experiment which was repeated 10 times.
Fig. 1. Effect of Ab and PrP106-126 on BMM survival. BMM (10 5 cells), from which CSF-1 had just been removed (day 0), were cultured for 3 days in RPMI / 10% FBS in the absence (unfilled) or presence of 50 mg / ml Ab1-42 (11 mM) (solid fill) or PrP 106-126 (26 mM) (shaded). Viable cell numbers (see Material and methods) from triplicate cultures were then measured. Data are mean values6S.E.M. from a typical experiment which was repeated five times. *P,0.001 and **P,0.004, compared to untreated group (Student’s t-test).
mM), and was not apparent at 3 mg / ml for both stimuli (data not shown). During the course of these studies, it was noted that some preparations of Ab1-42 and PrP106-126 gave a greater effect than others. We noted that the stock solutions (500 mg / ml) of both Ab1-42 and PrP106-126 formed aggregates on storage at 4 8C; in earlier studies we have reported that aggregated ox.LDL was more potent than ox.LDL in promoting BMM survival (and DNA synthesis (see below)) [15]. Consistent with the possibility that the degree of aggregation of the above peptides could be contributing to the batch-by-batch variation, we found that they were more active if the stock solutions were stored $2 weeks at 4 8C prior to use (data not shown); all of the data presented in this study was with peptides that had been stored for at least this period. Ab42-1 was inactive at 11 mM and the stock solution (500 mg / ml) did not form aggregates when stored under the same conditions as Ab1-42 (data not shown).
level than for an optimal CSF-1 concentration (5000 U / ml); in other words, the enhanced survival was associated with a small degree of DNA synthesis. As a control, Ab42-1 (50 mg / ml) was inactive (data not shown, but see Fig. 2). We then tested the PrP peptide, PrP178-193, which corresponds to a-helix 2 of PrP and which can aggregate into fibrils in culture [32]. It can be observed that PrP178193 was also able to induce DNA synthesis at a comparable level to Ab1-42. A similar activity was found for both Ab1-40 (data not shown) and PrP106-126 (Fig. 3).
3.3. Effect of Ab and PrP peptides on macrophage DNA synthesis in the presence of suboptimal CSF-1 CSF-1 circulates in vivo at low concentrations which are believed to be responsible mostly for the survival of macrophage-lineage cells [4]—it is therefore likely that many macrophage populations are exposed to such low CSF-1 concentrations in vivo and whose properties should
3.2. Effect of Ab and PrP peptides on macrophage DNA synthesis Several publications have shown that BMM can proliferate to CSF-1 in a dose-dependent manner with lower concentrations promoting survival (see, for example, Refs. [13,19,22,33,34]). Data for a CSF-1 concentration (5000 U / ml) which is optimal for proliferation [13,22,33,34] are given in Table 1. We therefore determined whether Ab could also stimulate BMM DNA synthesis in addition to promoting survival. As can be seen in Table 1, Ab1-42 was able to induce DNA synthesis albeit at a much lower
Fig. 2. BMM DNA synthesis induced Ab in the presence of a low CSF-1 concentration. BMM, from which CSF-1 had just been removed, were cultured in RPMI / 10% FBS, in the absence of CSF-1 or in the presence of CSF-1 (160 or 5000 U / ml). In addition they were untreated (unfilled), treated with Ab1-42 (50 mg / ml) (solid fill) or Ab42-1 (50 mg / ml) (shaded). Cultures were pulsed with [ 3 H]TdR at 24 h for a further 48 h before harvest. Data are mean values from triplicate cultures from a representative experiment which was repeated three times.
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4. Discussion
Fig. 3. BMM DNA synthesis induced by PrP106-126 in the presence of a low CSF-1 concentration. CSF-1-deprived BMM were cultured in RPMI / 10% FBS, in the absence or in the presence of CSF-1 (160 or 5000 U / ml). In addition, they were either untreated (unfilled), or treated with PrP106-126 (12.5 mg / ml) (shaded). Cultures were pulsed with [ 3 H]TdR at 24 h for a further 48 h before harvest. Data are mean values from triplicate cultures from a representative experiment which was repeated seven times.
therefore be studied under these conditions. It has previously been reported in many publications (see, for example Refs. [13–16,19,33,34]) that in vitro such CSF-1 concentrations keep all BMM alive and induce some DNA synthesis. The small DNA synthesis response of BMM to such a low CSF-1 concentration (160 U / ml) can be seen in Fig. 2—during the culture period, BMM numbers were maintained in the presence of this level of CSF-1 [33] (data not shown); the comparison to the response obtained with a CSF-1 dose that is optimal for DNA synthesis, namely 5000 U / ml [13–16,19,22,33] is provided. Previously, we reported for BMM DNA synthesis that a number of particulate materials, when added together with low CSF-1 concentrations, elicited a response which was more than additive [13–16]. In Fig. 2, it can be observed that Ab1-42 gave more than an additive DNA synthesis response in the presence of 160 U / ml CSF-1; as a control, Ab42-1 was again inactive by itself and did not potentiate the response to the low CSF-1 dose. The potentiation of DNA synthesis by Ab42-1 was not noted at the higher (i.e. optimal) concentration of CSF-1 (5000 U / ml). We next tested the DNA synthesis induced by PrP106126 in the presence of the same low CSF-1 concentration. In Fig. 3, it can be seen that PrP106-126 also gave a more than additive DNA synthesis response in the presence of 160 U / ml CSF-1; a ‘scrambled’ form of PrP106-126, which does not aggregate or polymerize and which is not neurotoxic [20], was inactive (data not shown).
Like BMM, there is evidence in the adult mouse that brain macrophages and some microglia also derive from bone marrow precursors [8]; CSF-1 circulates normally, most likely to maintain macrophage-lineage survival and, in this context, op / op (CSF-1-deficient) mice have reduced numbers of microglia, supporting a role for CSF-1 in microglia development [39]. As for BMM, CSF-1 induces microglial cell proliferation, migration and activation [11]. Both Ab and PrP have been reported to be mitogenic for microglial cells [2,5]. All of these similarities suggest that BMM, as a pure macrophage population capable of being generated easily in large numbers, are a useful in vitro model population to study brain macrophage and microglial cell development and function. We found above that, in the absence of CSF-1, aggregating, fibrillogenic forms of both Ab and PrP could enhance BMM survival at the doses tested. If occurring in vivo the enhanced survival of macrophage-lineage cells (macrophages and microglia) would be sufficient to lengthen their tenure in a lesion leading to more cells being present in brain (for the potential significance, see below). In other words, increased proliferation of such cells would not be necessary. It is likely that brain macrophage-lineage cells will be exposed normally to low CSF-1 concentrations in the steady state [39]. In AD brain, there is increased immunoreactivity for the CSF-1-receptor on microglia [1], neurons in AD show increased CSF-1 expression in proximity to Ab deposits, and CSF-1 levels in AD cerebrospinal fluid are fivefold greater than in controls [9]. Therefore a case can be made for their in vitro biology to be studied in the presence of CSF-1. For these reasons, we studied the DNA synthesis response of BMM to Ab and PrP when CSF-1 was also added to the cultures. Our results show for the first time that when macrophages are exposed to Ab or PrP they are able to proliferate more efficiently in the presence of suboptimal levels of CSF-1, i.e. those that normally provide a survival signal and / or a weak proliferative response in vitro [13–16,19,22,33,34]. These low CSF-1 levels are similar to circulating levels [4] and may also be similar to those found in the AD brain. If this potentiation were occurring in vivo with Ab- or PrP-loaded macrophages or microglia then it could be contributing to their increased numbers observed in AD lesions [36] or to the gliosis observed in prion disease [7], respectively. The increased numbers of macrophage lineage cells (macrophages and microglia) in brain as a result of the above proposed mechanisms (i.e. enhanced survival or proliferation) would mean that there are more cells available to produce inflammatory mediators, such as cytokines, proteinases, eicosanoids, etc. As mentioned, macrophage activation has been widely studied as a model for microglial activation by Ab and PrP [6] and both macrophages and
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microglial cells have been shown to produce inflammatory mediators in response to these stimuli [24,27]. The role of the inflammatory response in the pathogenesis of AD and prion diseases is not completely clear [37]. Epidemiological studies suggest that inflammation may contribute to AD progression since there might be a reduced risk in patients treated with anti-inflammatory drugs [30]. However, activated microglia might also play a role in the clearance of Ab deposits by phagocytosis, thus preventing their harmful effects [40,41], and has been proposed as the mechanism for the reduction in plaque burden following vaccination with aggregated Ab peptide [3,18,31]. Increased numbers of these cells in brain lesions resulting from enhanced survival and / or proliferation would contribute to this protective activity. The subsequent clearance of amyloidogenic peptide could then prevent continued survival and expansion of microglial cell populations by removing the responsible stimulus. Both Ab and PrP can aggregate and are only slowly degraded by macrophages / microglia [25,29]. At the active concentrations used in this paper, it has been reported by others that they are aggregated to some extent in the culture medium [21,23]. The increased potency observed upon storage of Ab could be due to such aggregation [28]. We have previously found that poorly degraded particles, namely ox.LDL [13,15], particulate adjuvants [14] and arthritogenic crystals [16], show similar effects when added to BMM in vitro. Again, with possible analogy, ox.LDL became more potent upon aggregation [15]. Such changes in macrophage lineage cells were proposed to be relevant to the development and function, respectively, of foam cells in atherosclerosis, of antigen presenting cells when exposed to certain adjuvants, and of macrophages in arthritic joints [13–16]. In this connection, it has been noted that the engorgement of microglia with undigested Ab is strikingly similar to the conversion of macrophages into foam cells [25]. Also, Ab1-42 microaggregate uptake is mediated by type A scavenger receptors on macrophages that also mediate internalization of modified LDL [26]. Binding of Ab to scavenger receptors has also been reported [10]. Thus Ab and PrP can be added to the list of poorly degraded aggregates capable of promoting macrophage survival and even DNA synthesis, with the latter response being particularly marked in the presence of suboptimal CSF-1 concentrations. As discussed above, subsequent activation of the more numerous microglia and macrophages may contribute further to the pathological processes in AD or prion diseases or play an important role in the clearance of the neurotoxic agents.
Acknowledgements This work was supported by grants, including a Senior Principal Research Fellowship (JAH), from the National
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Health and Medical Research Council of Australia. R. Sallay is thanked for typing the manuscript.
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